Choledocholithiasis is a common type of clinical gallstone that may require hepatobiliary surgery. Its incidence in China is approximately 8%-10%, which continues to increase [1]. Stones formed in the bile duct are called primary bile duct stones. Most gallstones are found in the gallbladder and enter the extrahepatic or intrahepatic bile ducts through the cystic duct, leading to choledocholithiasis and hepatolithiasis, or secondary bile duct stones. About 10% of patients with gallstones also suffer from bile duct stones [2]. Common clinical symptoms include pain in the upper right abdomen, accompanied by nausea and vomiting, and fever. Bile duct stones may cause long-term biliary obstruction, which can have serious, potentially life-threatening consequences, such as secondary cholangitis, pancreatitis, and obstructive jaundice [3]. Choledocholithiasis warrants immediate treatment upon diagnosis.

Yanglingquan (GB34) was first recorded in the Lingshu Jing of ancient Chinese acupuncture and moxibustion medicine. It dredges the liver, gallbladder, and meridians, activates the collaterals, relaxes tendons, and relieves pain. It has widely been used to combat many conditions, such as liver and gallbladder diseases and knee swelling and pain.

The conventional choledocholithiasis treatment involves removing the stones via surgery and relieving the biliary obstruction. However, clinical observation indicates that postoperative liver function and clinical symptoms naturally improve over time, and may repeat and worsen. [1]. Studies have shown that stimulating GB34 can promote gallbladder contraction and common bile duct dilatation, which promotes bile production and excretion and has an analgesic effect [4-6]. Therefore, the stimulation of GB34 during postoperative choledocholithiasis recovery is an effective adjunctive treatment. Moreover, acupoint catgut embedding (ACE) is a modern acupuncture treatment method that can prolong the sense of acupuncture, obtain lasting curative effects, and reduce distress [7].

Numerous studies have shown that bile acids are closely related to the formation of gallstones [8,9]. Bile acids can form micelles in the gallbladder, which can dissolve cholesterol in bile, thereby preventing the formation of cholesterol crystals and gallstones. The main cholesterol excretion pathway involves bile acids being converted into cholesterol by the liver and then excreted in feces. In cases of cholestasis, the proportion of sulfated bile acid pool increases, which increases the compounds’ water solubility, resulting in more effective renal clearance and reduced intestinal reabsorption [10]. Studies indicate that the oral intake of ursodeoxycholic acid (UDCA) can improve symptoms in patients with gallstones [11,12]. 3-oxocholic acid can be completely reduced to 3α-hydroxy bile acids via 3α-hydroxysteroid dehydrogenase [13].

In this study, we used the bile metabolomics method based on a high-resolution mass spectrometry to study the effects of ACE treatment at the GB34 acupoint on the bile composition of patients with choledocholithiasis. We also examined the correlations among differential metabolites, investigated six biochemical bile indices, and explored the molecular mechanism of ACE for choledocholithiasis treatment.

1. Patients

1) General information

This study was conducted in accordance with the declaration of Helsinki. The trial was reviewed and approved by the Ethics Committee of the Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine, number: JXFYLL2017103013. All subjects provided signed informed consent before participating in the trial.

2) Inclusion criteria

We recruited patients with choledocholithiasis from the general surgery ward at the Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine. The subjects met the diagnostic criteria for choledocholithiasis and were diagnosed with choledocholithiasis by ultrasonography and CT.

3) Exclusion criteria

We excluded individuals with other diseases from this study.

2. Methods

1) Sampling method

Patients only received routine postoperative antibiotic treatment. After the operation, we took bile samples before and after the ACE stimulation of GB34, for a total of 32 samples. On the fifth day after surgery, the patients fasted in the morning, and we collected their bile samples for an hour from 7 a.m. to 8 a.m. for biochemical analyses. At around 10 a.m., an ACE at bilateral GB34 acupoint was performed. The next morning, we similarly collected bile samples. After the biochemical tests, the bile samples were immediately stored in the refrigerator at –80℃.

2) Materials

We obtained methanol and acetonitrile from CNW Technologies (Germany), ammonium acetate from Sigma–Aldrich (Germany), and ammonium hydroxide from Fisher Chemical Company.

3) Metabolite extraction

We transferred a 50 μL bile sample into the EP tube. After adding 200 μL of extract solution (acetonitrile: methanol = 1:1, containing isotopically-labeled internal standard mixture), we vortexed the samples for 30 s, sonicated them for 10 min in an ice-water bath, and incubated them at –40℃ for 1 h to precipitate the proteins. Then, we centrifuged the samples at 12,000 rpm (RCF = 13,800 g, R = 8.6 cm) for 15 min at 4℃. We then transferred the supernatant to a fresh glass vial for analysis. We prepared the quality control (QC) samples by mixing equal volumes of the supernatants from all of the bile samples.

4) UHPLC-MS/MS analysis

We performed the LC-MS/MS analyses using a UHPLC system (Vanquish, Thermo Fisher Scientific) with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm) coupled with a Q-Exactive HFX mass spectrometer (Orbitrap MS, Thermo). Mobile phase A consisted of 25 mmol/L ammonium acetate and 25 mmol/L ammonia hydroxide in water (pH = 9.75), and mobile phase B consisted of acetonitrile. The gradient program was as follows: 0-0.5 min, 95% B; 0.5-7 min, 95%-65% B; 7-8 min, 65%-40% B; 8-9 min, 40% B; 9-9.1 min, 40%-95% B; and 9.1-12 min, 95% B. The flow rate was 0.5 mL/min and the column temperature was 30℃. The auto-sampler temperature was maintained at 4℃ and the injection volume was 3 μL. QC samples help to monitor instrument stability and reflect experimental reliability and data quality.

We used the QE HFX mass spectrometer to acquire MS/MS spectra in information-dependent acquisition (IDA) mode controlled by the acquisition software (Xcalibur, Thermo). In this mode, the acquisition software continuously evaluates the full scan MS spectrum. The ESI source conditions were set as follows: the sheath gas flow rate was 30 Arb, Aux gas flow rate was 25 Arb, the capillary temperature was 350℃, full MS resolution was 60,000, MS/MS resolution was 7,500, collision energy was 10/30/60 in the NCE mode, and the spray voltage was 3.6 kV (positive) or –3.2 kV (negative).

To ensure the system’s stability and reproducibility, we prepared the QC samples by mixing equal volumes of each bile sample and injecting every six samples during the batch analysis.

5) Data analysis

We converted the raw data into mzXML format using ProteoWizard and processed it using the anin-house program, which was developed using the R package and is based on XCMS for the detection, extraction, alignment, and integration of peaks [14]. We eliminated original peak data with missing values greater than 50% in all groups. The missing values in the data were filled with median values. After the data was initially grouped, we exported it to SIMCA 14.1 for principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLS-DA) analysis. Markers with VIP > 1 are screened based on the OPLS-DA mathematical model. We performed a one-way analysis of variance (one-way ANOV A) via SPSS 21.0 and used established tags (VIP > 1 and p < 0.1) to screen differentially labeled compounds. Then, we used an in-house MS2 database (BiotreeDB), MassBank (http://www.massbank.jp/), and Human Metabolome Database (HMDB, http://www.hmdb.ca/) to identify metabolites.

We performed all of the statistical analyses using IBM SPSS Statistics v21.0 (Chicago, USA). We compared the two groups using the student’s t-test, and analyzed the omics data using Pearson’s correlation analysis or Spearman correlation analysis. A p-value < 0.05 indicated a statistically significant difference.

1. Demographic information

From October 2017 to December 2020, we selected 16 eligible patients with choledocholithiasis (33-82 years of age, with an average age of 60 years.) The sample included seven men and nine women. Surgical treatment methods were adopted according to the patients’ medical conditions: (1) laparoscopic cholecystectomy + choledocholithotomy + T-tube drainage; (2) choledocholithotomy + T-tube drainage; and (3) left lateral liver resection + choledocholithotomy + T-tube drainage. No staff turnover or missing data were noted.

2. Quality control

We extracted three internal standard (IS) peaks for method verification. The relative standard deviation (RSD) values of the intensity are shown in Supplementary Tables 1 and 2. The IS response values in all QC samples showed good concordance (Supplementary Fig. 1). The correlation of QC samples close to 1 (correlation coefficient r ≥ 0.80) indicates good instrument stability and data quality during the detection process (Fig. 1)

Figure 1. Verification of metabolomic methodology: pearson’s correlation analysis of quality control samples (A) positive ion mode, (B) negative ion mode. The darker the color, the greater the correlation. The correlation is greater than 0.90, indicating that the whole detection process is stable, and the data quality is high.

3. Analysis of bile metabolomics before and after GB34 acupoint thread-embedding

The study subjects included 16 patients with bile duct stones who had undergone surgery and their bile samples before and after ACE at GB34 acupoint. A total of 32 bile samples were collected, which were divided into preoperative (Pre) and postoperative (Post) measurements (n = 16 in each measurement). Based on the QE quadrupole-electrostatic field orbitrap high-resolution mass spectrometry system, we analyzed the metabolic profiles of the bile samples in both the positive and negative ion modes. The total ion chromatograms (TIC) of the typical sample are shown in Supplementary Fig. 2.

The PLS-DA analysis showed changes in the participants’ metabolic profiles, indicating significant alterations in their physiology and body metabolic status (POS: R2X = 0.44, R2Y = 0.99, Q2 = 0.52. NEG: R2X = 0.27, R2Y = 0.95, Q2 = 0.60). The OPLS-DA model only considers the variables related to grouping to better differentiate the two measurements, select the differential metabolites, and verify the model reliability by the cross-validation graph of the permutation test (POS: R2X = 0.35, R2Y = 0.48, Q2 = –0.26. NEG: R2X = 0.61, R2Y = 0.18, Q2 = –0.29) (Fig. 2).

Figure 2. PLS-DA score plots of Pre and Post measurements (A) positive ion mode, (B) negative ion mode. OPLS-DA score plots of preoperative and postoperative (C) positive ion mode, (D) negative ion mode. Cross-validation plots of OPLS-DA model with 100 permutation tests (E) positive ion mode, (F) negative ion mode.

We used the OPLS-DA model to obtain the score plots in the positive and negative ion modes. The differential metabolites (VIP > 1) in the two measurements were obtained through their VIP value. Based on the HMDB, ChemSpider, MassBank, and related literature survey, we identified a total of six metabolic biomarkers in the positive ion mode and four metabolic biomarkers in the negative ion mode as significantly altered (VIP > 1, p < 0.05) via multivariate statistical analysis of bile samples from 16 patients (Table 1).

↑ = increased; ↓ = decreased; RT = retention time; EM = experimental mass; HMDB = human metabolome database..

&md=tbl&idx=1' data-target="#file-modal"">Table 1

Summary of the metabolites with significant changes in bile before and after acupoint catgut embedding treatment.

No.	RT (s)	Name	Formula	EM	Ion mode	HMDB	Levels	p-value
P1	86.4327	7-Dehydrocholesterol	C27H44O	453.3315496	[M+H+HCOONa]+	HMDB0000032	↑	0.027042
P2	127.714	DG (18:0/0:0/22:6n3)	C43H72O5	732.5528466	[M+ACN+H]+	HMDB0056061	↑	0.047775
P3	133.2255	PE (P-18:0/20:4[5Z,8Z,11Z,14Z])	C43H78NO7P	758.5673969	[M+Li]+	HMDB0005779	↓	0.036367
P4	212.793	Cholesterol sulfate	C27H46O4S	449.3089803	[M+H-H2O]+	HMDB0000653	↓	0.03235
P5	212.802	3-Oxocholic acid	C26H40O6	448.3051221	[M+ACN+H]+	HMDB0000502	↓	0.032311
P6	245.742	13’-Carboxy-alpha-tocopherol	C29H48O4	524.3709	[M+ACN+Na]+	HMDB0012555	↑	0.043801
P7	184.938	Succinic acid	C4H6O4	117.0186164	[M-H]–	HMDB0000254	↑	0.012307
P8	92.2585	Taurolithocholic acid 3-sulfate	C26H45NO8S2	280.6219908	[M-2H]–	HMDB0002580	↑	0.048282
P9	137.927	Xanthine	C5H4N4O2	197.0311692	[M+FA-H]–	HMDB0000292	↑	0.026292
P10	296.0605	3-Hydroxy-2-methylpyridine-4,5-dicarboxylate	C8H7NO5	218.0062176	[M+Na-2H]–	HMDB0006955	↓	0.042225

↑ = increased; ↓ = decreased; RT = retention time; EM = experimental mass; HMDB = human metabolome database..

As compared to the Pre measurement, 7-dehydrocholesterol, DG (18:0/0:0/22:6n3), 13’-Carboxy-alpha-tocopherol, succinic acid, taurolithocholic acid 3-sulfate, and xanthine increased significantly, while PE (P-18:0/20:4[5Z,8Z,11Z,14Z]), cholesterol sulfate, 3-Oxocholic acid, and 3-Hydroxy-2-methylpyridine-4,5-dicarboxylate decreased significantly in the Post measurement.

All 10 potential biomarkers were subjected to metabolic pathway analysis (MetPA) using the KEGG online database and MetaboAnalyst 5.0. The results indicate that the ACE at GB34 acupoint might affect the body’s metabolic pathways, including glycerophospholipid metabolism, steroid biosynthesis, the citrate cycle (TCA cycle), purine metabolism, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, butanoate metabolism, propanoate metabolism, alanine, aspartate and glutamate metabolism, and steroid hormone biosynthesis (Fig. 3).

Figure 3. Overview of metabolic pathway analysis. 1 = Glycerophospholipid metabolism. 2 = Steroid biosynthesis. 3 = 3Citrate cycle (TCA cycle). 4 = Purine metabolism. 5 = Glycosylphosphatidylinositol (GPI)-anchor biosynthesis. 6 = Butanoate metabolism. 7 = Propanoate metabolism. 8 = Alanine, aspartate and glutamate metabolism. 9 = Steroid hormone biosynthesis.

4. Pre-post (paired sample) t-test of the bile biomarkers and clinical liver function indices

To comprehensively analyze the metabolic differences in the bile samples, we also tested the biochemical indicators commonly used for hepatobiliary diseases, such as total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), total bile acid (TBA), total cholesterol (CHOL), calcium (CA), and one-hour bile flow reflecting the direct difference (Fig. 4). The results showed that the six clinical indicators had a downward trend after surgery, and the one-hour bile flow rate had an upward trend. Due to the small sample size, the trends were not significant.

Figure 4. Analysis of six biochemical indices of bile in the patients before and after GB34 point thread-embedding. (A) Total bilirubin (TBIL), (B) direct bilirubin (DBIL), (C) indirect bilirubin (IBIL), (D) total bile acid (TBA), (E) total cholesterol (CHOL), (F) calcium (CA), and (G) 1 h bile flow.

The present study showed that the ACE at the GB34 acupoint could alter the contents of bile acids in patients with choledocholithiasis, including 3-oxocholic acid and taurolithocholic acid 3-sulfate. The contents of sulfate bile acid increased after ACE, suggesting that the stimulation of GB34 could reverse the metabolic disorder caused by cholestasis, which changed the bile acid metabolism and increased bile acid sulfate levels. These results illustrated the intervention effect of ACE treatment on bile acid metabolism. Studies have shown that healthy people have a specific bile microbiota, and patients with gallstones are significantly different [15]. The production and metabolism of 3-oxycholic acid are associated with certain intestinal bacteria [16,17]. This suggests the involvement of intestinal bacteria in this interference process.

Cholesterol sulfate is an important steroid sulfate that plays important physiological roles in the human body. Cholesterol sulfate participates in cholesterol biosynthesis by inhibiting sterol synthesis at the 3-hydroxy-3-methylglutaryl-CoA reductase level [18]. It also participates in steroid synthesis [19], inhibits T cell receptor signals [20], and stabilizes the cell membrane [21]. This study suggests that the reduction of cholesterol sulfate might regulate the biosynthesis and metabolism of cholesterol in patients with bile duct stones. We determined the CHOL content in the bile, which suggested a downward trend after ACE.

ACE treatment could also regulate the glycerol and phospholipids in bile. The bile lipids are composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and a small amount of sphingomyelin; PC accounts for more than 95% of the bile lipids [22]. PC is almost the only phospholipid in normal human bile. Its synthesis begins with the combination of diacylglycerol and phosphatidic acid choline. In the liver, PE can be converted into PC by the addition of 3 N-methyl measurements [23]. Micelles formed by bile salts and phospholipids help dissolve the cholesterol in bile. In addition to increasing the solubility of cholesterol in bile, phospholipids help prevent bile toxicity in hepatobiliary and gastrointestinal epithelial cells [24].

The present study aimed to identify relevant pathways to elucidate the mechanism by which ACE can be used to treat choledocholithiasis. To the best of our knowledge, the effects of ACE at the GB34 acupoint on bile metabolism in patients with bile duct stones have not been previously studied. The Pre and Post measurements had differing levels of metabolites. Although there was no normal control measurement as a reference, the analysis of bile metabolites before and after ACE could explain the effects of stimulating the GB34 acupoint.

This study reported the changes in bile metabolites among patients with bile duct stones treated by ACE at GB34 to further analyze possible mechanisms. However, the small sample size might limit the study results. Future studies are needed to obtain more clinical samples and should involve serum metabolomics and gut microbial analyses.

In conclusion, ACE treatment at the GB34 acupoint helps regulate bile acid, cholesterol, and phospholipid metabolism in the bile of patients with choledocholithiasis. Future studies should focus on metabolic changes, serum metabolomics, and an intestinal microbial analysis.

Supplementary data to this article can be found online at https://doi.org/10.51507/j.jams.2024.17.4.133.

This study was supported by the National Key R&D Program of China (2017YFC1702902 and 2019YFC1712300), Jiangxi university of traditional Chinese medicine 1050 youth talent project, and Jiangxi University of Chinese Medicine Science and Technology Innovation Team Development Program.

H.F. and X.J. designed the research. X.J. and D.L. performed the experiment. X.R., Q.M., H.D., H.O., Y.Z. and L.L. analyzed data. L.L. and H.F. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

The authors declare no conflict of interest.